A potential influenza virus pandemic caused by the possible crossover of the highly pathogenic H5 avian influenza virus poise a great threat to the public health. Although current influenza vaccines are effective in eliciting protective immunity in young healthy adults, their efficacy in young children and elderly adults is less adequate. Therefore, the development of novel vaccine strategies that can induce more potent protective immune responses, especially in these high-risk populations, is in urgent need. Our group has been focused on developing strategies to enhance immune responses induced by DNA vaccines as well as VLP (virus-like particle) vaccines. Recently, we found that immunization with a mixture of DNA and VLP (DNA/VLP) vaccines induced both antibody and cellular immune responses at high levels in immunized animals, indicating that a combined benefit of the two vaccine platforms can be obtained with this novel vaccination strategy. In this exploratory study, we propose to apply this novel approach for developing more potent influenza vaccines, with the aim to induce strong antibody and cellular immune responses at both mucosal and systemic compartments. We plan to achieve our goals through carrying out the studies outlined in the following specific aims. Specific Aim 1. To test the hypothesis that DNA/VLP vaccines (a mixture of DNA and VLP vaccines) are more effective in eliciting protective immune responses against influenza virus infection. We will produce VLPs comprised of the influenza virus A/PR8 M1 and HA proteins as well as DNA vaccines expressing the HA protein, and compare immune responses induced by immunization with DNA/VLP vaccines to the immune responses induced by immunization with individual VLP or DNA vaccines as well as inactivated influenza viruses. Furthermore, to investigate the potential advantage of the novel DNA/VLP vaccination strategy in aged populations, we will compare immune responses induced in aged mice by different vaccine preparations as well as their abilities to confer protection against challenge by lethal doses of influenza viruses. Specific Aim 2. To test the hypothesis that simultaneous mucosal and systemic immunization with DNA/VLP vaccines will elicit strong immune responses against influenza at both mucosal and systemic compartments and confer more effective protection. Induction of strong immune responses at both mucosal and systemic compartments is particularly important for protection against influenza infection. We will compare immune responses induced by DNA/VLP vaccines delivered via alternative immunization strategies, with the aim to select an optimal approach that can induce strong antibody and cellular immune responses at both mucosal and systemic compartments, for achieving a more effective control of influenza virus infection and facilitate virus clearance. [unreadable] [unreadable] [unreadable]